The aldol addition reaction is a reversible reaction involving the combination of two reactant molecules and the formation of a product having a new carbon-carbon bond. Each of the reactants contains a carbonyl group, i.e., either an aldehyde or ketone. During the reaction, one of the reactants loses a proton from the carbon atom next to its carbonyl group, thereby becoming nucleophilic. The nucleophilic carbon of the first reactant then attacks the carbonyl group of the second reactant. The reverse of this condensation reaction can also occur and entails the cleavage of a carbon-carbon bond and the dissociation of a molecule into two components. The aldol addition reaction is important in the glycolytic pathway and is catalyzed by aldolase enzymes. The aldol addition reaction is also fundamental to organic chemistry for the formation and dissociation of carbon-carbon bonds. In organic chemistry, the reaction may be catalyzed by base.
Two mechanistic classes of aldolase enzymes have evolved, viz., Class I and Class II aldolases. (W. J. Rutter, Fed. Proc. Amer. Soc. Exp. Biol. (1964): vol. 23, p 1248.) Class I aldolases utilize the .epsilon.-amino group of a Lys in the active site to form a Schiff base with one of the substrates, which activates the substrate as an aldol donor.
The mechanism for class I aldolases is illustrated in FIG. 1. The reaction is bimolecular and proceeds through covalent catalysis through multiple intermediates. An iminium ion or Schiff base forms that acts as an electron sink, which lowers the activation energy (E.sub.a) for proton abstraction from C.alpha. and subsequent enamine formation. The enamine acts as the carbon nucleophile, or aldol donor, which reacts with an aldehyde electrophile, the aldol acceptor, to form a new C--C bond. The Schiff base is then hydrolyzed and the product is released. The essence of the mechanism is the formation of the enamine which is the nascent carbon nucleophile.
Class II aldolases are metalloenzymes that facilitate enolate formation by coordination to the substrate's carbonyl oxygen. Transition state models have also been disclosed for aldol reactions involving metals. (H. E. Zimmerman et al., J. Am. Chem. Soc. (1957): vol. 79, p 1920.) However, the mechanism for Class II aldolases remains to be fully characterized.
A number of enzymes catalyze the aldol condensation. The mechanisms of these enzymes have been well characterized. (C. Y. Lai, et al., Science (1974): vol. 183, p 1204; and A. J. Morris et al., Biochemistry (1994) vol. 33, p 12291.) However, aldolase enzymes accept a relatively limited range of substrates (C. -H. Wong et al., Enzymes in Synthetic Organic Chemistry (Permagon, Oxford, 1994); M. D. Bednarski in Comprehensive Organic Synthesis, B. M. Trost, Ed.(Pergamon, Oxford, 1991), vol 2, pp. 455-473; C. F. Barbas III, et al., J. Am. Chem. Soc. (1990): vol 112, p 2013; H. J. M. Gijsen et al., J. Am. Chem. Soc. (1995): vol. 117, p 2947; C. -H. Wong et al., J. Am. Chem. Soc. (1995): vol. 117, p. 3333; L. Chen, et al., J. Am. Chem. Soc (1992): vol. 114, p 741.) Although natural aldolase enzymes display broad specificity with respect to the aldol acceptor, the aldol donor is usually limited to the natural substrate. The art of organic synthesis would benefit significantly if catalysts having the desired substrate specificity could be produced to order for catalyzing desired aldol addition reactions.
Non-enzymic base catalyzed aldol addition reactions are employed widely in organic chemistry to form new carbon-carbon bonds. Also, a variety of effective reagents have been developed to control the stereochemistry of the aldol. However, these reagents are stoichiometric and require pre-formed enolates and extensive protecting group chemistry. (C. H. Heathcock, Aldrichim. Acta (1990): vol. 23, p 99; C. H. Heathcock, Science (1981): vol. 214, p 395; D. A. Evans, Science (1988): vol. 240, p 420; S. Masamune, et al., Angew. Chem. Int. Ed. Engl. (1985): vol. 24, p 1; D. A. Evans, et al., Top. Stereochem. (1982): vol. 13, p 1; C. H. Heathcocket et al., in Comprehensive Organic Synthesis, B. M. Trost, Ed. (Pergamon, Oxford, 1991), vol. 2, pp. 133-319 (1991); and I. Paterson, Pure & Appl. Chem. (1992): vol. 64, 1821.) Recently catalytic aldol reactions that use pre-formed enolates have been developed, including the Mukaiyama cross-coupling aldol. (S. Kobayashi, et al., Tetrahedron (1993): vol. 49, p 1761; K. Furuta, et al., J. Am. Chem. Soc. (1991): vol. 113, p 1041; T. Bach, Angew. Chem. Int. Ed. Engl. (1994): vol. 33, p 417 and references therein; and E. M. Carreira, et al., J. Am. Chem. Soc. (1995): vol. 117, p 3649.)
For some reactions, the problem of complex intermediates may be solved by using relatively reactive compounds rather than the more usual inert antigens to immunize animals or select antibodies from libraries such that the process of antibody induction involves an actual chemical reaction in the binding site. (C. F. Barbas III, et al., Proc. Natl. Acad. Sci. USA (1991): vol. 88, p 7978 (1991); K. D. Janda et al., Proc. Natl. Acad. Sci. USA (1994): vol. 191, p 2532.) This same reaction then becomes part of the catalytic mechanism when the antibody interacts with a substrate that shares chemical reactivity with the antigen used to induce it.
One of the major goals of organic chemistry is to use the understanding of reaction mechanisms to design new catalysts. This is often not easy because one must address intermediates that are of high energy and complex structure. Antibody catalysts offer one potential solution to this problem in that they can be programmed by the experimenter to interact with the rate limiting transition state of a chemical reaction thereby lowering its energy and increasing the reaction rate. (R. A. Lerner, et al., Science (1991): vol. 252, p 659.) However, even here the ability of the experimenter to program the catalyst is usually limited to the more global aspects of the transition state rather than the detailed reaction mechanism. Thus, while one can deal with high energy charges, stereoelectronic, and geometrical features that appear along the reaction coordinate, the organization of multiple complex reaction intermediates remains difficult.
What is needed is a method for inducing antibodies that use the reaction mechanisms that give aldolases their efficiency but that take advantage of the range of substrates and stereochemical specificities available with antibodies. What is need is a strategy which would amalgamate the best features of the simple chemical and enzymatic approaches to the problem of forming carbon-carbon bonds via the aldol condensation which is, arguably, the most basic C--C bond forming reaction in chemistry and biology.